Modulation of expression of LDH isoenzymes in endothelial cells by laminin: implications for angiogenesis

Endothelial cell (EC) matrix interaction is critical in angiogenesis. Although matrix components can regulate the process of angiogenesis by acting as a reservoir of various cytokines, it is not clear if extracellular matrix (ECM) can modulate the production and activity of angiogenic cytokines. Investigations were therefore carried out to study the influence of the basement membrane (BM) protein, laminin (Ln) on the activity of vascular endothelial growth factor (VEGF), the major angiogenic cytokine, using isolated human umbilical vein ECs (HUVECs) in culture. Analysis of the biochemical markers of angiogenesis confirmed proangiogenic effect of Ln. The levels of VEGF protein and mRNA were not different in cells maintained on Ln, collagen I or polylysine substrata. Chorioallantoic membrane assay using VEGF isolated from cell extracts however revealed that Ln increased its angiogenic potency. Immunoblotting and HPLC analysis showed considerable reduction in poly adenosyl ribosylation of VEGF associated with a significant decrease in the levels of NAD+, in cells maintained on Ln substrata. Further, a shift in the isoenzymic pattern of LDH towards the B rich forms and an upregulation of LDH B gene were observed in cells maintained on Ln. Ln modulates expression of LDH gene through alpha(6)beta(4) integrin mediated downstream signaling involving p38 mitogen activated protein kinases (MAPK) pathway. It thus appears that Ln can affect aerobic metabolism of ECs by modulating the expression of LDH isoenzymes resulting in a decrease in the level of NAD+ that can cause a reduction in the poly adenosyl ribosylation of VEGF altering its angiogenic potency.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Visual deprivation suppresses L5 pyramidal neuron excitability by preventing the induction of intrinsic plasticity

In visual cortex monocular deprivation (MD) during a critical period (CP) reduces the ability of the deprived eye to activate cortex, but the underlying cellular plasticity mechanisms are incompletely understood. Here we show that MD reduces the intrinsic excitability of layer 5 (L5) pyramidal neurons and enhances long-term potentiation of intrinsic excitability (LTP-IE). Further, MD and LTP-IE induce reciprocal changes in K(v)2.1 current, and LTP-IE reverses the effects of MD on intrinsic excitability. Taken together these data suggest that MD reduces intrinsic excitability by preventing sensory-drive induced LTP-IE. The effects of MD on excitability were correlated with the classical visual system CP, and (like the functional effects of MD) could be rapidly reversed when vision was restored. These data establish LTP-IE as a candidate mechanism mediating loss of visual responsiveness within L5, and suggest that intrinsic plasticity plays an important role in experience-dependent refinement of visual cortical circuits.     

Serum paraoxonase-1 (PON1) activities (PONase/AREase) and polymorphisms in patients with type 2 diabetes mellitus in a North-West Indian population

Objective

Paraoxonase-1 (PON1), an HDL-C associated enzyme, protects lipoproteins from oxidation. There is evidence that PON1 enzyme activity is reduced in the patients with type 2 diabetes mellitus (T2DM). North-West Indian Punjabis, a distinct ethnic group has high incidence of T2DM. However till date there is no information regarding PON1 enzyme activities and PON1 polymorphisms in T2DM patients of this ethnic group.

Methods

We identified polymorphisms in the coding Q192R, L55M and promoter − 909G/C, − 162A/G, − 108C/T of the PON1 gene by using PCR-RFLP, multiplex PCR and allele specific oligonucleotide PCR assays in 250 T2DM patients and 300 healthy controls. We also assessed paraoxonase (PONase) and arylesterase (AREase) activities of PON1 enzyme.

Results

The serum PONase (114.2 vs. 178.0 nmol/min/ml) and AREase (62.7 vs. 82.5 μmol/min/ml) activities were significantly lower (p < 0.0001) in patients as compared to controls. PONase activity was affected by all the studied PON1 polymorphisms. However, AREase activity was not affected by any of these polymorphisms. Coding Q192R and promoter − 909G/C polymorphisms showed significant differences in genotypic distribution. QR, RR (Q192R) and GC, CC (− 909G/C) genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes showed significant association with type 2 diabetes. No significant linkage disequilibrium was observed among the five polymorphisms.

Conclusion

Both PONase and AREase activities are lower in patients and this could lead to increased lipid peroxidation and accelerated atherosclerosis in them. PONase activity, but not AREase activity is influenced by PON1 polymorphisms. QR, RR, GC, CC genotypes and L-C-A-R-G, L-T-A-R-G, L-T-G-Q-C haplotypes are commoner in diabetics as compared to controls and may be related to genetic susceptibility to type 2 diabetes.     

Adenine recognition is a key checkpoint in the energy release mechanism of phage T4 DNA packaging motor

ATP is the source of energy for numerous biochemical reactions in all organisms. Tailed bacteriophages use ATP to drive powerful packaging machines that translocate viral DNA into a procapsid and compact it to near-crystalline density. Here we report that a complex network of interactions dictates adenine recognition and ATP hydrolysis in the pentameric phage T4 large "terminase" (gp17) motor. The network includes residues that form hydrogen bonds at the edges of the adenine ring (Q138 and Q143), base-stacking interactions at the plane of the ring (I127 and R140), and cross-talking bonds between adenine, triphosphate, and Walker A P-loop (Y142, Q143, and R140). These interactions are conserved in other translocases such as type I/type III restriction enzymes and SF1/SF2 helicases. Perturbation of any of these interactions, even the loss of a single hydrogen bond, leads to multiple defects in motor functions. Adenine recognition is therefore a key checkpoint that ensures efficient ATP firing only when the fuel molecule is precisely engaged with the motor. This may be a common feature in the energy release mechanism of ATP-driven molecular machines that carry out numerous biomolecular reactions in biological systems.     

Direct Observation of Treatment Provided by a Family Member as Compared to Non-Family Member among Children with New Tuberculosis: A Pragmatic,Non-Inferiority,Cluster-Randomized Trial in Gujarat,India

Background

The World Health Organization recommends direct observation of treatment (DOT) to support patients with tuberculosis (TB) and to ensure treatment completion. As per national programme guidelines in India, a DOT provider can be anyone who is acceptable and accessible to the patient and accountable to the health system, except a family member. This poses challenges among children with TB who may be more comfortable receiving medicines from their parents or family members than from unfamiliar DOT providers. We conducted a non-inferiority trial to assess the effect of family DOT on treatment success rates among children with newly diagnosed TB registered for treatment during June–September 2012.

Methods

We randomly assigned all districts (n = 30) in Gujarat to the intervention (n = 15) or usual-practice group (n = 15). Adult family members in the intervention districts were given the choice to become their child’s DOT provider. DOT was provided by a non-family member in the usual-practice districts. Using routinely collected clinic-based TB treatment cards, we compared treatment success rates (cured and treatment completed) between the two groups and the non-inferiority limit was kept at 5%.

Results

Of 624 children with newly diagnosed TB, 359 (58%) were from intervention districts and 265 (42%) were from usual-practice districts. The two groups were similar with respect to baseline characteristics including age, sex, type of TB, and initial body weight. The treatment success rates were 344 (95.8%) and 247 (93.2%) (p = 0.11) among the intervention and usual-practice groups respectively.

Conclusion

DOT provided by a family member is not inferior to DOT provided by a non-family member among new TB cases in children and can attain international targets for treatment success.

Trial Registration

Clinical Trials Registry–India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2015/09/006229     

Protective effects of vitamin E against atrazine-induced genotoxicity in rats

Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) is one of the most commonly used herbicides to control grasses and weeds. The widespread contamination and persistence of atrazine residues in the environment has resulted in human exposure. Vitamin E is a primary antioxidant that plays an important role in protecting cells against toxicity by inactivating free radicals generated following pesticides exposure. The present study was undertaken to investigate the protective effect of vitamin E against atrazine-induced genotoxicity. Three different methods: gel electrophoresis, comet assay and micronucleus test were used to assess the atrazine-induced genotoxicity and to evaluate the protective effects of vitamin E. Atrazine was administered to male rats at a dose of 300 mg/kg body weight for a period of 7, 14 and 21 days. There was a significant increase (P<0.001) in tail length of comets from blood and liver cells treated with atrazine as compared to controls. Co-administration of vitamin E (100 mg/kg body weight) along with atrazine resulted in decrease in tail length of comets as compared to the group treated with atrazine alone. Micronucleus assay revealed a significant increase (P<0.001) in the frequency of micronucleated cells (MNCs) following atrazine administration. In the animals administrated vitamin E along with atrazine there was a significant decrease in percentage of micronuclei as compared to atrazine treated rats. The increase in frequency of micronuclei in liver cells and tail length of comets confirm genotoxicity induced by atrazine in blood and liver cells. In addition, the findings clearly demonstrate protective effect of vitamin E in attenuating atrazine-induced DNA damage.     

Swim exercise training and adaptations in the antioxidant defense system of myocardium of old rats: relationship to swim intensity and duration

We examined a suitable swim program of different intensities and durations that could evoke changes in the myocardial antioxidant capacity in 22-month-old rats. Male rats (Rattus norvegicus) were assigned to either a sedentary control (SE-C) group or one of six trainee groups. Animals were swim-exercised for 4 weeks with either 20 min or 40 min/day, and three intensities, low, moderate and high. Low-intensity at 20 min/day elicited maximum swim velocity (Sv) and endurance capacity (P<0.05). While serum total cholesterol, triglyceride and low-density lipoprotein (LDL-C) levels were significantly reduced, high-density lipoprotein (HDL-C) showed an increase (P<0.05) in low-intensity trained rats (20 min/day) over SE-C. Notable reduction in blood lactate was also evident. Exercise training significantly increased superoxide dismutase (Mn-SOD), decreased lipid peroxidation products, malondialdehyde and lipofuscin in the left and right ventricles. Increased Mn-SOD with concomitant decrease in lipofuscin in left ventricle was significantly greater than in right ventricle. Moderate- to high-intensity exercise was not effective in either reducing lipid peroxidation products or elevating Mn-SOD activity. These data suggest that swim training at low-intensity of 20 min/day is beneficial as a major protective adaptation against oxidative stress in old myocardium.